WO2019156152A1 - Acide nucléique comprenant une séquence de régulation de l'expression des gènes dépendant de l'arabinose - Google Patents

Acide nucléique comprenant une séquence de régulation de l'expression des gènes dépendant de l'arabinose Download PDF

Info

Publication number
WO2019156152A1
WO2019156152A1 PCT/JP2019/004378 JP2019004378W WO2019156152A1 WO 2019156152 A1 WO2019156152 A1 WO 2019156152A1 JP 2019004378 W JP2019004378 W JP 2019004378W WO 2019156152 A1 WO2019156152 A1 WO 2019156152A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleotide sequence
promoter
nucleic acid
sequence
arabinose
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2019/004378
Other languages
English (en)
Japanese (ja)
Inventor
修平 中根
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Green Earth Institute Co Ltd
Original Assignee
Green Earth Institute Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Green Earth Institute Co Ltd filed Critical Green Earth Institute Co Ltd
Priority to JP2019570794A priority Critical patent/JP7374475B2/ja
Publication of WO2019156152A1 publication Critical patent/WO2019156152A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium

Definitions

  • the present invention relates to a nucleic acid sequence that enables gene expression control depending on arabinose or an analog thereof in coryneform bacteria. More specifically, the present invention relates to a nucleic acid comprising a gene expression control sequence having a nucleotide sequence that can function as a promoter in a coryneform bacterium and an AraR binding sequence comprising a predetermined nucleotide sequence.
  • coryneform bacteria typified by the same species have chemicals including various amino acids, organic acids, alcohols including ethanol, etc. Widely used in the production of goods.
  • coryneform bacteria basic research such as genome analysis and gene expression analysis has been vigorously conducted, and gene recombination techniques for coryneform bacteria have been almost established.
  • the types of chemicals that can be produced by coryneform bacteria are increasing, and mass production of these chemicals is also becoming possible. .
  • the use of coryneform bacteria in the production of chemicals and useful substances is gaining increasing attention in the industry.
  • Patent Document 1 An example of a promoter that functions in a coryneform bacterium is a technique using a promoter sequence of an aspartase-encoding gene isolated from Brevibacterium flavum MJ-233 strain.
  • Patent Document 1 only the negative control plasmid without the promoter sequence was introduced in the above strain into which the reporter plasmid containing the predetermined promoter sequence and the chloramphenicol acetyltransferase (CAT) gene was introduced. It is described that CAT activity 25 times higher than that of the strain was confirmed.
  • CAT chloramphenicol acetyltransferase
  • Patent Document 2 some DNA fragments isolated from the genomic DNA of Brevibacterium flavum strain MJ-233, which exhibit a promoter function stronger than the tac promoter in coryneform bacteria, are known.
  • Patent Document 2 since the promoter activity of these DNA fragments depends on the composition of the carbon source (various sugars, ethanol, protein degradation products, etc.) of the medium in which the coryneform bacterium serving as the host is cultured, It is described that the expression of the target gene can be controlled by changing the carbon source composition.
  • P EF-TU the promoter of EF-TU
  • P sod the promoter of superoxide dismutase gene
  • Pgro a promoter of GroES gene
  • PEF-TS EF-TS promoter
  • Patent Document 7 a gene expression technique using the respective promoter sequences of Cgl1565 gene (locus NCgl1504) and Cgl1360 gene (locus NCgl1305) derived from Corynebacterium glutamicum ATCC13032 strain is also known (Patent Document 7).
  • a plasmid construct is constructed by introducing each of the above promoter sequences and a predetermined reporter gene or enzyme gene, and a Corynebacterium glutamicum transformant into which the plasmid construct is introduced is obtained.
  • promoter activity was recognized for each of the above promoter sequences by reporter assay using the transformant or measurement of enzyme activity.
  • Patent Document 8 describes an expression cassette or vector using a promoter sequence derived from Brevibacterium ammoniagenes CJHB100 strain. These promoter sequences are promoter sequences identified by culturing the same strain, identifying proteins that are overexpressed in each culture face, and cloning the 5 ′ untranslated region of the genes encoding those proteins. In Patent Document 7, a total of seven promoter sequences named pcj1 to pcj7 were obtained, and the promoter activity of these promoter sequences was evaluated by a reporter assay using the GFP gene in Brevibacterium ammoniagenes.
  • Patent Document 7 confirms that these promoter sequences can also function in Escherichia coli ( Escherichia coli ).
  • pcj1 exhibits high activity in both coryneform bacteria and Escherichia coli. Has been suggested.
  • the promoter sequences disclosed in Patent Documents 1 to 8 are wild-type promoter sequences of coryneform bacteria
  • mutant promoter sequences whose promoter activity has been improved by introducing mutations to the wild-type promoter sequences are also available.
  • mutant promoter sequences include the diaminopimelate dehydrogenase (ddh) gene, LysC-asd operon gene, and aspartate aminotransferase (aspB) gene derived from Corynebacterium glutamicum.
  • mutant promoters obtained by introducing predetermined mutations Patent Documents 9 to 11.
  • a series of techniques relating to promoters described in Patent Documents 9 to 11 aim to improve the amount of lysine produced by increasing the expression level of an enzyme gene involved in the lysine biosynthetic pathway.
  • Patent Document 12 includes two or more promoter sequences of P EF-TU , P sod , P gro , and P EF-TS derived from Corynebacterium glutamicum described in Patent Documents 3 to 6. Multiple promoters linked in tandem are disclosed, and the results of improved expression of target genes in multiple promoters are also shown compared to the case where each of these promoters is used alone.
  • Patent Document 13 uses a promoter sequence obtained by comprehensively analyzing the gene promoter of Corynebacterium glutamicum that is promoted or suppressed under anaerobic conditions.
  • a promoter sequence capable of promoting or suppressing gene expression under anaerobic conditions is used, various kinds of substances necessary for target substance production are produced when producing target substances under anaerobic conditions using coryneform bacteria. It is suggested that gene expression can be improved and expression of various genes unnecessary for target substance production can be suppressed, so that more efficient target substance production can be achieved. .
  • expression systems that use the functions available heterologous promoter in Corynebacterium glutamicum has been known for a long time, is in such expression systems, e.g. tac, trc, LacUV5, P R , P L , etc. Escherichia coli expression system using the above (for example, see Non-Patent Documents 1 to 3).
  • expression of promoter activity is induced by addition of an inducer such as lactose or its analog isopropylthiogalactoside (IPTG).
  • a heat-inducible expression vector in which a ⁇ PL promoter and latent promoters CJ1 and CJ4 isolated from Corynebacterium ammoniagenes are combined.
  • This heat-inducible expression vector is Corynebacterium ammonia. It has been confirmed that it functions not only in Genes but also in Corynebacterium glutamicum (Non-Patent Document 4).
  • Non-Patent Document 5 describes that the expression of the target gene could be strictly controlled without showing non-specific non-basal expression. Furthermore, ⁇ -ketoglutarate dehydrogenation in coryneform bacteria using this expression system. It is described that when the expression of the odhI gene encoding an enzyme inhibitor was regulated, high levels of glutamic acid production could be realized.
  • Non-Patent Document 6 discloses that, in Corynebacterium glutamicum ATCC31831 strain, AraR protein, which is a LacI type transcriptional regulator, has an araBDA gene responsible for L-arabinose catabolism, an araE gene responsible for arabinose absorption, and It has been suggested to suppress the expression of the transcription regulatory factor AraR gene.
  • Non-Patent Document 6 also shows the results of evaluation of the galM and araR promoter regions by the LacZ reporter assay. For the AraR promoter region, cells cultured with D-glucose added and L-arabinose added. No difference in promoter activity was observed between cells cultured in this manner, and that the expression level of LacZ increased in response to the addition of L-arabinose for the galM promoter.
  • Non-Patent Documents 1 to 4 that controls expression of a target gene by exposing a recombinant coryneform bacterium under a predetermined stress environment such as an expression inducer such as IPTG or temperature,
  • a predetermined stress environment such as an expression inducer such as IPTG or temperature
  • IPTG or temperature a predetermined stress environment
  • problems of toxicity and cost of the inducer and complicated expression control process of the target gene and a certain level of non-specific expression can be seen even under the base where expression is not induced.
  • the control of expression is often difficult.
  • the growth of coryneform bacteria is inhibited by such non-specific expression under the base.
  • Non-Patent Document 5 AraC gene derived from E. coli, the gene expression system that combines P BAD promoter and AraE gene effect confirmation test using a coryneform bacterium by the present inventors As a result, about 10 times as much promoter activity was observed when arabinose was added as compared with arabinose not added, but no significant promoter activity as described in Non-Patent Document 5 was observed. That is, it cannot be said that the gene expression system described in Non-Patent Document 5 has poor reproducibility and can withstand practical use.
  • Non-Patent Document 5 uses a promoter sequence or gene derived from Escherichia coli as described above, and the gene expression system according to the present invention is a nucleotide sequence and an amino acid. The configuration is completely different at the array level.
  • Non-Patent Document 6 in Corynebacterium glutamicum ATCC31831, the AraR protein suppresses the expression of the araBAD gene, the araE gene and the transcription regulator AraR gene, and the AraR protein is a gene of the arabinose gene group. Although it is suggested that it can bind to a predetermined sequence in the interstitial region, it does not describe a specific configuration of a gene expression control system using these gene groups and AraR protein binding sequences at a feasible level. . Actually, as shown in Comparative Test Example 1 of the Example below, the present inventor confirmed the promoter region of the araE gene including the AraR protein binding sequence.
  • Non-Patent Document 6 is an academic paper that merely shows the analysis results regarding the role of AraR protein in the arabinose gene expression regulatory mechanism of Corynebacterium glutamicum. It does not imply availability.
  • an object of the present invention is to provide a gene expression system capable of strictly controlling the expression of a target gene in coryneform bacteria and ensuring a good expression level when inducing the expression of the target gene.
  • a gene expression control sequence (R) having at least a part of a nucleotide sequence (X) capable of functioning as a promoter and the nucleotide sequence (Y) described in the following (a) or (b):
  • the nucleotide sequence (X) is trc promoter, tacI promoter, tacII promoter, T5 promoter, T7 promoter, lac promoter, trp promoter, tet promoter, EFtu promoter, groES promoter, SOD promoter, P15 promoter, ldhA promoter, gapA
  • the nucleotide sequence (X) infects a promoter sequence derived 5 ′ upstream of a gene present in the genomic DNA of a coryneform bacterium, a promoter sequence derived from a plasmid inherent in the coryneform bacterium, or a coryneform bacterium.
  • the gene expression control sequence (R) shows a promoter activity that is at least 10 times higher in coryneform bacteria when arabinose is added than when arabinose is not added.
  • the gene expression control sequence (R) exhibits a promoter activity that is at least 15 times higher in coryneform bacteria when arabinose is added than when arabinose is not added. [1] to [1] [6] The nucleic acid according to any one of [6]. [8] The gene expression control sequence (R) exhibits a promoter activity that is at least 20 times higher when arabinose is added in coryneform bacteria than when arabinose is not added. [1] to [1] [7] The nucleic acid according to any one of [7].
  • the gene expression control sequence (R) shows a promoter activity that is at least 35 times higher in coryneform bacteria when arabinose is added than when arabinose is not added. [8] The nucleic acid according to any one of [8]. [10] The gene expression control sequence (R) shows a promoter activity that is at least 50 times higher in coryneform bacteria when arabinose is added than when arabinose is not added. [1] to [1] [9] The nucleic acid according to any one of [9]. [11] The gene expression control sequence (R) shows a promoter activity that is at least 70 times higher in coryneform bacteria when arabinose is added than when arabinose is not added. [1] to [1] [10] The nucleic acid according to any one of [10].
  • nucleic acid according to any one of [1] to [11], wherein the nucleotide sequence (Y) is represented by the following general formula (I): 5′-N 1 TGTN 2 AGCGN 3 TN 4 AN 5 N 6 N 7 -3′—General formula (I)
  • N 1 , N 2 , N 3 , N 4 , N 5 , N 6 and N 7 each independently represent A (adenine), G (guanine), C (cytosine) or T (thymine).
  • T (thymine) should be read as U (uracil).
  • nucleic acid according to any one of [1] to [12], wherein the gene expression control sequence (R) comprises any one of the following nucleotide sequences (1) to (o): (L) the nucleotide sequence set forth in any one of SEQ ID NOs: 22 to 61 and SEQ ID NOs: 96 to 102; (M) a nucleotide sequence in which one or more bases have been deleted, substituted or added in the nucleotide sequence according to (l); (N) a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence that is complementary to the nucleotide sequence according to (l); and (o) at least 80% or more of the nucleotide sequence according to (l) Nucleotide sequences having identity, However, when the nucleic acid is RNA, thymine (t) in the base sequence is replaced with uracil (u).
  • the SD sequence is directly or indirectly linked to the 3 ′ end of the nucleotide sequence (Y).
  • nucleic acid according to any one of [1] to [16], comprising a nucleotide sequence encoding at least one of an araE protein and an araR protein derived from a coryneform bacterium.
  • nucleic acid comprising a nucleotide sequence encoding at least one of an araE protein and an araR protein derived from a coryneform bacterium.
  • nucleic acid comprising a nucleotide sequence encoding at least one of an araE protein and an araR protein derived from a coryneform bacterium.
  • R gene expression control sequence
  • the nucleic acid according to any one of [1] to [21] which is DNA.
  • the following bacteria are provided.
  • the bacterium according to [23] which is an Escherichia bacterium.
  • DNA having at least one nucleotide sequence encoding each of araE protein and araR protein derived from coryneform bacteria is incorporated into genomic DNA, and said genomic DNA can express said araE protein and araR protein
  • the expression method of the following target genes is provided.
  • a method for expressing a target gene comprising exposing the bacterium according to any one of [23] to [26] to arabinose or an analog thereof to express the target gene.
  • a method for producing a target substance which comprises expressing the target gene by exposing the bacterium according to any one of [23] to [26] to arabinose or an analog thereof.
  • nucleic acid fragments for use in controlling expression of a target gene in coryneform bacteria, A nucleic acid fragment comprising the nucleotide sequence (Y) described in the following (a) or (b): (A) the nucleotide sequence set forth in any one of SEQ ID NOs: 5 to 9, 11, and 12 and SEQ ID NOs: 17 and 18; (B) a nucleotide sequence in which one or more bases have been deleted, substituted or added in the nucleotide sequence described in (a) above,
  • the nucleic acid is RNA
  • thymine (t) in the nucleotide sequence shall be read as uracil (u)
  • the nucleotide sequence shown in each of SEQ ID NOs: 10, 20, and 21 as the nucleotide sequence (Y) is excluded, and the nucleotide sequence (Y) satisfies the following condition (II)
  • efficient and precise gene expression control can be performed, and an efficient bioprocess can be provided.
  • the expression suppression state of the target gene is released and the expression of the target gene can be promoted, so that the operation and work in the bioprocess becomes easy.
  • FIG. 4 is a diagram schematically showing the structure of a plasmid vector constructed in Test Example 3.
  • A) shows the structure of plasmid vector pGE837, and
  • B) shows the structure of plasmid vector pAra1. It is a figure which shows the result of the reporter assay implemented in Test Example 3.
  • A shows the structure of the plasmid vector pGE728-1 constructed in Test Example 1
  • b shows the structure of the plasmid vector pGE716 constructed in Test Example 3.
  • FIG. 4 is a diagram schematically showing the structure of a plasmid vector constructed in Test Example 3.
  • A) shows the structure of plasmid vector pGE837, and
  • (b) shows the structure of plasmid vector pAra1. It is a figure which shows the result of the reporter assay implemented in Test Example 3.
  • not added indicates a sample without arabinose added
  • added indicates a sample with arabinose added
  • induction rate indicates a promoter activity value / sample without arabinose added with the arabinose added sample. The ratio of the promoter activity value (fold) is shown. It is a figure which shows the result of the reporter assay implemented in Test Example 3.
  • not added refers to a sample without arabinose added. It is a schematic diagram showing the promoter region of the araE gene in Corynebacterium glutamicum ATCC31831 genomic DNA.
  • the “nucleic acid” according to the present invention is a nucleic acid for use in controlling expression of a target gene in bacteria including coryneform bacteria.
  • the nucleic acid according to the present invention may be provided in any form of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid).
  • the nucleic acid according to the present invention may be in a single-stranded or double-stranded form.
  • the nucleic acid is specifically an isolated nucleic acid, cDNA or cRNA.
  • the nucleic acid of the present invention is DNA.
  • RNA Ribonucleic acid
  • the predetermined gene expression control function of the present invention is exerted.
  • it may be provided in the form of RNA.
  • Techniques for converting RNA into DNA using reverse transcriptase and the like are known to those skilled in the art.
  • the nucleic acid may be subjected to chemical modification such as methylation.
  • the nucleic acid according to the present invention may be any nucleic acid as long as the gene expression control sequence (R) satisfies the above (I) when promoter activity is measured by a reporter assay or the like as described later.
  • the nucleic acid may or may not have a nucleotide sequence encoding the target gene.
  • the nucleic acid according to the embodiment that does not include the nucleotide sequence encoding the target gene is specifically an expression plasmid construct (nucleotide sequence encoding the target gene) to be used for expression of the target gene in bacteria including coryneform bacteria. Is an expression cassette or expression vector (that is, before inserting a target gene into a predetermined cloning site).
  • the “coryneform bacterium” refers to a group of microorganisms defined in Barges Manual of Detergent Bacteriology, Vol. 8, p. 599, 1974. More specifically, the coryneform bacteria include Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, Micrococcus. ) Genus, Microbacterium genus and the like.
  • Corynebacterium glutamicum for example, FERM P-18976 strain, ATCC13032 strain, ATCC31831 strain, ATCC13058 strain, ATCC13059 strain, ATCC13060 strain, ATCC13232 strain, ATCC13286 strain, ATCC13345 strain, ATCC13345 strain, ATCC13345 strain, ATCC13345 strain, ATCC13345 strain ATCC13761 strain, ATCC14020 strain); Corynebacterium acetoglutamicum (eg ATCC 15806 strain); Corynebacterium acetoacidophilum (eg, ATCC 13870 strain); Corynebacterium melasscola (eg ATCC 17965 strain); Corynebacterium efficiens ; Corynebacterium alkanolyticum (for example, ATCC 21511 strain); Corynebacterium callunae (eg ATCC 15991 strain); Corynebacterium lil
  • Corynebacterium herculis for example, ATCC 13868 strain.
  • Corynebacterium ammoniagenes (Corynebacterium Sutationisu) (Corynebacterium ammoniagenes (Brevibacterium ammoniagenes) ( for example ATCC6871 shares, ATCC6872 shares).
  • Brevibacterium divaricatum eg, ATCC 14020 strain
  • Brevibacterium flavum eg, MJ-233 (FERM BP-1497) strain, MJ-233AB-41 (FERM BP-1498) strain, ATCC 13826 strain, ATCC 14067 strain, ATCC 13826 strain
  • Brevibacterium immariophyllum eg, ATCC 14068 strain
  • Brevibacterium lactofermentum Corynebacterium glutamicum
  • Brevibacterium lactofermentum (Corynebacterium glutamicum)
  • Brevibacterium lactofermentum (Corynebacterium glutamicum)
  • Brevibacterium roseum eg, ATCC 13825 strain
  • Brevibacterium saccharolyticum eg, ATCC 14066 strain
  • Brevibacterium thiogenitalis eg, ATCC 19240 strain
  • Brevibacterium album eg ATCC 15111 strain
  • Brevibacterium cerinum for example, ATCC 15
  • Arthrobacter include the following species and strains.
  • Arthrobacter globiformis for example, ATCC 8010 strain, ATCC 4336 strain, ATCC 21056 strain, ATCC 31250 strain, ATCC 31338 strain, ATCC 35698 strain
  • ATCC 8010 strain for example, ATCC 8010 strain, ATCC 4336 strain, ATCC 21056 strain, ATCC 31250 strain, ATCC 31338 strain, ATCC 35698 strain
  • Micrococcus fredenreichii [for example, No. 239 (FERM P-13221) strain]; Micrococcus luteus [e.g. 240 (FERM P-13222) strain]; Micrococcus ureae (for example, IAM1010 strain); Micrococcus roseus (for example, IFO3764 strain) and the like.
  • Micrococcus fredenreichii [for example, No. 239 (FERM P-13221) strain]
  • Micrococcus luteus [e.g. 240 (FERM P-13222) strain]
  • Micrococcus ureae for example, IAM1010 strain
  • Micrococcus roseus for example, IFO3764 strain
  • microbacterium ammoniaphilum for example, ATCC 15354 strain.
  • the ATCC is an abbreviation for American Type Culture Collection (PO Box 1549 Manassas, VA 20108 USA), and the above ATCC stocks can be sold by the same organization. .
  • the coryneform bacterium may be a wild-type strain that originally exists in nature, or a set in which genomic DNA or plasmid DNA is manipulated or a predetermined gene or artificial sequence is introduced into these DNAs. It may be a substitute. Further, the coryneform bacterium may be a mutant produced by exposure to a predetermined chemical substance or environmental condition.
  • nucleotide sequence (X) capable of functioning as a promoter in coryneform bacteria refers to a nucleotide sequence exhibiting promoter activity in any one or more of the species / strains belonging to coryneform bacteria as described above. Point to.
  • the nucleotide sequence (X) may be an artificially synthesized sequence or a natural sequence present in an organism including bacteria.
  • nucleotide sequence (X) capable of functioning as a promoter in coryneform bacteria means that RNA polymerase specifically binds in coryneform bacteria and initiates transcription to mRNA by the transcription activity of the RNA polymerase.
  • RNA polymerase specifically binds in coryneform bacteria and initiates transcription to mRNA by the transcription activity of the RNA polymerase.
  • operator sequences for example, lacO
  • ribosome binding sequences (Shine-Dalgarno sequence; SD sequence), etc.
  • promoter refers to only a nucleotide sequence that can purely initiate transcription into mRNA by the transcription activity of RNA polymerase.
  • nucleic acid according to the present invention is not intended to exclude the presence of additional gene regulatory sequences as described above.
  • the nucleotide sequence (X) is not particularly limited as long as it can function as a promoter in any one or more of strains / strains belonging to coryneform bacteria.
  • an embodiment that employs a nucleotide sequence that exhibits promoter activity in common for all species belonging to coryneform bacteria is preferred.
  • a nucleotide sequence that can function as a promoter in the genus Escherichia eg, Escherichia coli
  • the nucleotide sequence (X) is a promoter sequence derived from the 5 ′ upstream region of a gene endogenous to bacterial genomic DNA, including coryneform bacteria as described above, autonomous replication in these bacteria.
  • the promoter sequences derived from these bacteria and phages may be wild-type sequences, or may be those obtained by introducing a predetermined mutation into the wild-type sequences.
  • the promoter sequence is located 5 ′ upstream of the transcription start point and is known to be characterized by a ⁇ 35 box and a ⁇ 10 box. Therefore, the primer extension method, quantitative RT-PCR, etc. It is also possible to determine the transfer start point by the known method, and obtain the transfer start point in consideration of the determined transfer start point information. That is, in the present invention, a known promoter sequence may be used as the nucleotide sequence (X), but the present invention is not particularly limited thereto, and a promoter sequence newly obtained as described above may be used, or an artificial sequence may be used. Alternatively, a newly created promoter may be used.
  • the types of promoter sequences that can be used as the nucleotide sequence (X) in the present invention include an inducible promoter that can induce expression of a target gene by adding specific culture conditions or chemical substances, and RNA polymerase. Examples thereof include a constitutive promoter that depends on availability and enables constant expression.
  • the type of promoter used as the nucleotide sequence (X) is not particularly limited, but is preferably a constitutive promoter. This is because when the nucleotide sequence (X) can function as a constitutive promoter, the nucleotide sequence (X) can be used under the absence of arabinose due to the presence of the predetermined nucleotide sequence (Y) of the present invention. ) Can be strictly suppressed, and the suppression state of the promoter activity can be released by a simple operation of adding arabinose, so that significant expression of the target gene can be promptly induced.
  • the nucleotide sequence (X) may be a promoter of an enzyme gene involved in various amino acid biosynthesis systems in bacteria including coryneform bacteria or a derivative thereof (mutation introduction sequence). More specifically, glutamate biosynthesis enzyme genes (eg, glutamate dehydrogenase genes), glutamine synthesis enzyme genes (eg, glutamine synthase genes), lysine biosynthesis enzyme genes (eg, aspartokinase genes), threonine biosynthesis Synthetic genes (eg homoserine dehydrogenase gene), isoleucine and valine biosynthesis enzyme genes (eg acetohydroxy acid synthase gene), leucine biosynthesis enzyme genes (eg 2-isopropylmalate synthase gene), proline and Arginine biosynthesis enzyme genes (eg glutamate kinase gene), histidine biosynthesis enzyme genes (eg phosphoribosyl-ATP pyrophosphorylase gene), aromatic amino acid biosynthesis systems such as tryptophan,
  • the nucleotide sequence (X) may be a promoter of a gene encoding a factor involved in cell division, such as a divIVA gene promoter.
  • a promoter that operates in the logarithmic growth phase as a nucleotide sequence (X) is also preferably used, for example, divIVA, gap, ldhA, fda, glyA, cysK, aroF, gpmA, eno, fumC, pfk, sdhA, mdh, argF
  • Examples include promoters of various genes such as proA, proC, aceE, serA, metE, nifS1, tpi, aceD, cysD, sdhB, and pck.
  • nucleotide sequence (X) strong promoters used in E. coli expression systems such as trc promoter, tac promoter, T5 promoter, T7 promoter, lac promoter, trp promoter, tet promoter and the like are also preferably used.
  • the nucleotide sequence (X) is disclosed in EF-Ts promoter, EF-Tu promoter, groES promoter, SOD promoter, P15 promoter, gapA promoter, dapA promoter, tuf promoter, metE promoter, and Patent Documents 1 to 13.
  • Various promoters can also be used.
  • the gene expression control sequence (R) in the nucleic acid according to the present invention includes the nucleotide sequence (Y) described in the above (a) or (b) in addition to the nucleotide sequence (X).
  • nucleotide sequence described in (a) is the nucleotide sequence described in any one of SEQ ID NOs: 5 to 12. These nucleotide sequences are derived from the AraR protein binding sequence present on the genomic DNA of Corynebacterium glutamicum. Their nucleotide sequences are shown below.
  • nucleic acid adopting the nucleotide sequence described in any one of SEQ ID NOs: 5 to 12 and SEQ ID NOs: 17 and 18 as a nucleotide sequence (Y) was introduced into a coryneform bacterium.
  • a nucleic acid adopting the nucleotide sequence described in any one of SEQ ID NOs: 5 to 12 and SEQ ID NOs: 17 and 18 as a nucleotide sequence (Y) was introduced into a coryneform bacterium.
  • nucleotide sequences of (a) and (b) are preferred.
  • nucleotide sequence (Y) in order to realize the gene expression control effect at a more remarkable level, it is even more preferable to employ the nucleotide sequence defined in (a) above as the nucleotide sequence (Y). It is still more preferable to employ the nucleotide sequence shown in any of (i) to (vi) above, and a higher effect can be expected as going from (i) to (vi).
  • nucleotide sequence (Y) is defined in the above (b) on the assumption that the gene expression control sequence (R) satisfies the condition (I) in addition to the nucleotide sequence defined in the above (a).
  • Nucleotide sequences can also be employed. That is, the nucleotide sequence defined in (b) is a nucleotide sequence described in any one of SEQ ID NOs: 5 to 12 and SEQ ID NOs: 17 and 18, wherein one or more bases are deleted, substituted, or added. Nucleotide sequence.
  • the range of “one or more” is not particularly limited as long as the gene expression control sequence (R) satisfies the condition (I).
  • the range is, for example, 1 to 10, 1 to 9, 1 to 8, preferably 1 to 7, more preferably 1 to 6, even more preferably 1 to 5, particularly preferably 1 to There can be four, one to three, one to two, or one.
  • nucleotide sequence (Y) is a nucleotide sequence represented by the following general formula (I). 5′-N 1 TGTN 2 AGCGN 3 TN 4 AN 5 N 6 N 7 -3′—General formula (I)
  • N 1 , N 2 , N 3 , N 4 , N 5 , N 6 and N 7 each independently represent A (adenine), G (guanine), C (cytosine) or T (thymine).
  • N 1 is preferably A (adenine) or G (guanine)
  • N 2 is preferably G (guanine) or T (thymine)
  • N 3 is A (adenine)
  • C N 4 is preferably A (adenine), C (cytosine) or G (guanine)
  • N 5 is A (adenine) or C (cytosine).
  • N 6 is preferably A (adenine), C (cytosine) or T (thymine)
  • N 7 is preferably C (cytosine) or T (thymine).
  • T (thymine) is read as U (uracil).
  • any position (for example, 1 to 3 positions or 1 to 2 positions) of N 1 to N 7 nucleotides is deleted in the nucleotide sequence represented by the general formula (I).
  • a nucleotide sequence can be employed as the nucleotide sequence (Y).
  • a nucleotide sequence in which one or both of N 2 and N 3 are deleted is a nucleotide sequence (Y). May be adopted.
  • the positional relationship between the nucleotide sequence (X) and the nucleotide sequence (Y) is not particularly limited as long as the gene expression control sequence (R) satisfies the condition (I).
  • the nucleotide sequence (Y) may be linked directly or indirectly to the 5 ′ end of the nucleotide sequence (X), or the nucleotide sequence (Y) is linked directly or indirectly to the 3 ′ end of the nucleotide sequence (X). May be.
  • the nucleotide sequence (Y) may exist in an untranslated region or an intergenic region, or may exist in a region (open reading frame) encoding a predetermined gene.
  • nucleotide sequence (X) and at least a part of the nucleotide sequence (Y) overlap can be included in the present invention.
  • Specific examples of embodiments in which at least a part of the nucleotide sequence (X) and at least a part of the nucleotide sequence (Y) overlap each other include the following.
  • nucleotide sequence (Y) i) a form in which the entire nucleotide sequence (Y) is encompassed within the nucleotide sequence (X) region; ii) a form in which the 3 ′ part of the nucleotide sequence (Y) overlaps with the 5 ′ part of the nucleotide sequence (X); and iii) A form in which the 5 ′ part of the nucleotide sequence (Y) overlaps with the 3 ′ part of the nucleotide sequence (X).
  • nucleotide sequences (X) and (Y) are not particularly limited, but the nucleotide sequence (Y) may be converted into the nucleotide sequence (X) in order to reliably produce the predetermined effect. ) Is preferably directly or indirectly linked to the 3 ′ end.
  • the nucleotide sequence (Y) is indirectly linked to the nucleotide sequence (X)
  • the 3 ′ end or 5 ′ end of the nucleotide sequence (Y) is the 5 ′ end or 3 ′ of the nucleotide (Y)
  • a phrase such as “indirectly linked to the end” means that the nucleotide sequence (X) and the nucleotide sequence (Y) are linked via a sequence consisting of one nucleotide or a plurality of nucleotides.
  • the number of nucleotides in the “sequence consisting of a plurality of nucleotides” is not particularly limited as long as the gene expression control sequence (R) satisfies the condition (I).
  • the “sequence consisting of a plurality of nucleotides” linking the nucleotide sequence (X) and the nucleotide sequence (Y) may be a simple linker sequence not having a specific function, or may be a further promoter sequence Further, it may be a sequence having a gene expression regulation function such as a ribosome binding sequence (Shine-Dalgarno sequence; SD sequence), other transcriptional regulatory sequences and translational regulatory sequences.
  • nucleotide sequence (Y) is directly linked to the nucleotide sequence (X)
  • the 3 ′ end or 5 ′ end of the nucleotide sequence (Y) is the 5 ′ end or 3 ′ end of the nucleotide (Y)
  • the phrase “directly linked to” may be understood literally, and the nucleotide sequence (X) and the nucleotide sequence (Y) do not go through one or more other nucleotides as described above. Means directly connected to
  • each of the nucleotide sequence (X) and the nucleotide sequence (Y) may be present one by one or plural in the nucleic acid of the present invention.
  • the plurality of nucleotide sequences (X) or nucleotide sequences (Y) have exactly the same sequence as each other. Or may have a different sequence.
  • condition (I) will be described.
  • the significance of satisfying the condition (I) by the gene expression control sequence (R) in the present invention is as follows.
  • the gene expression control sequence (R) includes the nucleotide sequence (X) functioning as a promoter for controlling the expression of the target gene and the AraR binding sequence present in the genomic DNA of the coryneform bacterium. Is included as a nucleotide sequence (Y).
  • the AraR protein binds to the nucleotide sequence (Y) in the coryneform bacterium into which the predetermined nucleic acid of the present invention has been introduced in an environment where the arabinose concentration is below the basal level. As a result, it is presumed that the promoter activity of the nucleotide sequence (X) is suppressed. However, when the coryneform bacterium is exposed to an environment in which the arabinose concentration exceeds the basal level, the AraR protein is subjected to allosteric regulation by arabinose and dissociated from the nucleotide sequence (Y).
  • the promoter activity of X) is induced and the expression of the target gene is promoted. That is, when the expression of the target gene is controlled using the nucleic acid according to the present invention, the phenomenon actually observed is that the promoter activity by the nucleotide sequence (X) is suppressed in an environment where the arabinose concentration is below the basal level. Thus, the expression of a predetermined gene under the control of the promoter activity of the nucleotide sequence (X) is suppressed at the level of transcription into mRNA or protein expression. On the other hand, in an environment where the arabinose concentration exceeds the basal level, the expression of the predetermined gene is promoted at the level of mRNA transcription or protein expression.
  • the gene expression control sequence (R) has the condition (I) that in the coryneform bacterium, the promoter activity of the nucleotide sequence (X) is suppressed when arabinose is not added compared to when arabinose is added. It is necessary to satisfy Here, the sufficiency of the condition (I) can be confirmed by a reporter assay shown below.
  • a DNA fragment obtained by linking a nucleotide sequence encoding a reporter gene to the 3 ′ end of the gene expression control sequence (R) configured as described above is introduced into a predetermined site of an expression vector that can function in coryneform bacteria.
  • the reporter gene include ⁇ -galactosidase gene (LacZ), ⁇ -glucuronidase, chloramphenicol acetyltransferase, various fluorescent proteins (eg, green fluorescent protein), and the like.
  • a nucleotide sequence encoding a reporter gene is directly or indirectly linked to the 3 ′ end of the gene expression control sequence (R), such as an expression vector plasmid having the nucleotide sequence shown in SEQ ID NO: 80 or 81.
  • R gene expression control sequence
  • a reporter plasmid may be constructed independently, or various reporter plasmid systems commercially available for reporter assays may be utilized. Then, a transformant in which such a DNA construct is introduced into coryneform bacteria is obtained.
  • the transformant is cultured for a certain period of time using a medium containing arabinose at a predetermined concentration or more and a medium containing arabinose less than a predetermined concentration, and the culture solution is sampled.
  • mRNA of the reporter gene is quantified by a technique such as quantitative PCR, or the activity of the reporter protein expressed from the reporter gene is appropriately measured.
  • the sufficiency of condition (I) can be determined by determining how many times the expression level of mRNA or reporter protein activity measured for the former culture solution sample is measured for the latter culture solution sample. it can.
  • the sufficiency of the condition (I) can be confirmed by, for example, a reporter assay method shown in the following examples. That is, in the plasmid vector pGE728-1 (SEQ ID NO: 80) used in Test Example 1 of the Examples below, a promoter sequence (PtacI) and an AraR binding sequence existing upstream of the reporter gene ⁇ -galactosidase gene (LacZ) By exchanging the region (SEQ ID NO: 32) formed by ligation with the gene regulatory sequence (R) to be evaluated for the sufficiency of the condition (I), and performing a reporter assay using the resulting plasmid vector, It is convenient to confirm the sufficiency of the condition (I) of the gene regulatory sequence (R).
  • a reporter assay method shown in the following examples. That is, in the plasmid vector pGE728-1 (SEQ ID NO: 80) used in Test Example 1 of the Examples below, a promoter sequence (PtacI) and an AraR binding sequence existing
  • assay conditions including conditions for induction by addition of arabinose, medium conditions, culture conditions such as culture temperature and culture time, etc. are not particularly limited as long as a reliable ratio of promoter activity can be obtained.
  • the assay conditions may be appropriately adjusted in consideration of the nature of the gene expression control sequence (R) to be selected, the type and nature of the reporter gene to be used, the nature of the coryneform bacterium to be used, and the like.
  • ⁇ -galactosidase gene (LacZ) assay is described in, for example, J. Org. Biol. Chem. 1995, 270: 11811-11189 or Genetics, 2010, 185: 823-830. A specific procedure is shown below as an example.
  • liquid medium 4% glucose, 25 ⁇ g / mL kanamycin
  • Each of the above cultures is cultured for a predetermined time, the cultures with and without arabinose are sampled, OD (610 nm) is measured, and 20 ⁇ L of 80 ⁇ L of permeation buffer (100 mM Na 2 HPO 4 , 20 mM KCl, 2 mM MgSO 4 , 0.8 mg / mL CTAB, 0.4 mg / mL sodium deoxycholate, 5.4 ⁇ L / mL ⁇ mercaptoethanol).
  • permeation buffer 100 mM Na 2 HPO 4 , 20 mM KCl, 2 mM MgSO 4 , 0.8 mg / mL CTAB, 0.4 mg / mL sodium deoxycholate, 5.4 ⁇ L / mL ⁇ mercaptoethanol.
  • a substrate solution (60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 1 mg / mL ONPG, 2.7 ⁇ L / mL ⁇ mercaptoethanol) is mixed with 600 ⁇ L and incubated at 25 ° C.
  • the absorbance (A420) of the supernatant is measured.
  • a mirror unit (Miller Unit) is calculated as the promoter activity.
  • 1 mirror unit 1000 ⁇ (A420 / t ⁇ V ⁇ OD610)
  • Formula (II) In the formula, t represents time (minutes), and V represents a culture solution (mL).
  • the culture medium which has the composition shown below, for example can be used.
  • the promoter activity value measured for the sample in which the promoter activity of the nucleotide sequence (X) was induced by the addition of arabinose or the like was measured for the control sample without arabinose added.
  • the meaning of “inducing the promoter activity by adding arabinose or the like” is only arabinose.
  • nucleotide sequence (Y) when another gene expression control factor (for example, lacO) is used, control by the other gene expression control factor in addition to arabinose addition is possible. This means that the necessary operations are required.
  • the gene expression control sequence (R) is, paradoxically, a promoter whose promoter activity value measured for a control sample without arabinose added is measured for a sample in which the promoter activity of the nucleotide sequence (X) is induced.
  • the activity value is preferably 1/5 or less, in this case, more preferably 1/10 or less, still more preferably 1/15 or less, 1/20 or less, 1/25 or less, 1/30 or less, 1 / 35 or less, 1/40 or less or 1/45 or less, particularly preferably 1/50 or less, 1/55 or less, 1/60 or less, 1/65 or less, 1/70 or less, 1/80 or less, 1/90 or less 1/100 or less, or 1/150 or less.
  • the sample without arabinose added The measured promoter activity is preferably 10 mirror units or less.
  • the upper limit of the promoter activity measured for a sample without arabinose added is preferably 9, more preferably 8, 7, 6, 5, 4, particularly preferably 3.5, 3.4, 3.3. 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2 0.0, 1.9, 1.8, 1.7, 1.6, 1.2, 1.1, 1.0 (units are mirror units).
  • a gene expression control sequence comprising any one of the following nucleotide sequences (p) to (s) can be employed.
  • P the nucleotide sequence set forth in any one of SEQ ID NOS: 22 to 41;
  • Q a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence according to (p);
  • R a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence that is complementary to the nucleotide sequence according to (p); and (s) at least 80% or more of the nucleotide sequence according to (p) Nucleotide sequences having identity, However, when the nucleic acid is RNA, thymine (t) in the nucleotide sequence is read as uracil (u).
  • SEQ ID NOs: 22 to 41 Each nucleotide sequence shown in SEQ ID NOs: 22 to 41 is shown below.
  • a gene expression control sequence containing any one of the following nucleotide sequences (t) to (s) can also be employed.
  • T the nucleotide sequence according to any one of SEQ ID NOs: 42 to 61;
  • U a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence described in (t);
  • V a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence that is complementary to the nucleotide sequence described in (t); and (w) at least 80% or more of the nucleotide sequence described in (t) Nucleotide sequences having identity, However, when the nucleic acid is RNA, thymine (t) in the nucleotide sequence is read as uracil (u).
  • SEQ ID NOs: 42 to 61 The nucleotide sequences shown in SEQ ID NOs: 42 to 61 are shown below.
  • SEQ ID NO: 43 (2-3): 5′-TTGACAATTTAATCATCGAACTAGTTTAATGTGTGGA ATGTGAGCGCTAACAC- 3 ′
  • the underlined sequence corresponds to the nucleotide sequence (Y), and the other 5 ′ upstream region is a nucleotide sequence. It corresponds to (X).
  • the nucleotide sequences (Y) in the gene expression control sequences (R) shown in (1-1) to (1-20) correspond to the nucleotide sequences shown in SEQ ID NOs: 5 to 12 and SEQ ID NOs: 17 and 18, respectively.
  • the nucleotide sequences (Y) in the gene expression control sequences (R) shown in (2-1) to (2-20) also correspond to the nucleotide sequences shown in SEQ ID NOs: 5 to 12 and SEQ ID NOs: 17 and 18, respectively.
  • the nucleotide sequence (X) in the sequences shown in (1-1) to (1-10) is a PtacI -35 box, -10 box and transcription frequently used in an expression system in Escherichia coli (E. coli). It is a core sequence including the start site (+1) and is a sequence considered necessary and sufficient for expression of promoter activity (Li et al. Microbial Cell Factories 2012, 11:19).
  • the nucleotide sequence (X) in the gene regulatory sequence (R) shown in (1-11) to (1-20) has a transcription start site (+1) in the PtacI sequence generally recognized by those skilled in the art. ) From the entire upstream region, and 6 nucleotides are further added to the 5 ′ end of the core sequence (Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 21). -25, January 1983).
  • PtacI SEQ ID NO: 1 or 2
  • R gene expression control sequence
  • nucleotide sequence (X) it is also preferable to use not only PtacI but also PtacII (SEQ ID NO: 3 or 4) as the nucleotide sequence (X).
  • PtacI SEQ ID NO: 3 or 4
  • the nucleotide sequences shown in the above (t) to (w) are included.
  • R gene expression control sequence
  • the nucleotide sequence (X) in the sequence shown in (2-1) to (2-10) defined in (t) above is the PtacII ⁇ 35 box often used in the expression system in Escherichia coli (E. coli).
  • nucleotide sequence (X) in the sequences shown in (2-11) to (2-20) is a sequence including the entire region upstream from the transcription start site (+1) in the PtacII sequence generally recognized by those skilled in the art. (6 nucleotides are further added to the 5 ′ end of the core sequence), and high promoter activity can be expressed in the same manner as PtacI (see the above-mentioned document).
  • a gene expression control sequence including any one of the following nucleotide sequences (h) to (k) can also be employed.
  • H the nucleotide sequence set forth in any one of SEQ ID NOs: 96 to 102;
  • I a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence according to (h);
  • J a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence that is complementary to the nucleotide sequence according to (h); and
  • Each of the nucleotide sequences shown in SEQ ID NOs: 96 to 102 is, as a nucleotide sequence (X), a tuf promoter, a dapA promoter, a metE promoter, a ldhA promoter, a gapA promoter, sod, which are present on the genome of Corynebacterium glutamicum ATCC 13031, respectively. It includes the nucleotide sequence of the promoter and the tuf promoter, and the nucleotide sequence shown in SEQ ID NO: 5 as the nucleotide sequence (Y).
  • nucleotide sequence according to SEQ ID NO: 102 is obtained by changing the linking site of the AraR binding sequence to the tuf promoter with respect to the nucleotide sequence according to SEQ ID NO: 96, which also employs the tuf promoter.
  • the putative transcription region 5 ′ terminal site is overlapped with the 5 ′ terminal region of the AraR binding sequence.
  • nucleotide sequence in the nucleotide sequence according to any one of SEQ ID NOs: 96 to 102 (h) ( Implementation including various modifications in which the region of X) (that is, the nucleotide sequence portion according to SEQ ID NO: 5) is substituted with the nucleotide sequence described in any one of SEQ ID NO: 6 to 12 and SEQ ID NOs: 17 and 18 A form (V) is mentioned.
  • the range of “one or more” is defined by the gene expression control sequence (R).
  • the range is, for example, 1 to 40, 1 to 35, 1 to 30, 1 to 30, 1 to 25, 1 to 20, 1 to 18, 1 to 15, 1 to 12 1 to 10, preferably 1 to 9, 1 to 8, 1 to 7, more preferably 1 to 6, even more preferably 1 to 5, particularly preferably 1 to 4, 1 to There can be three, one to two, or one.
  • nucleotide sequence hybridizing under stringent conditions is specifically Specifically, in the nucleic acid hybridization method such as the colony hybridization method, the plaque hybridization method, or the Southern blot hybridization method, the nucleotide sequence described in (p), (t) or (h) is used as a standard. A nucleotide sequence that forms a complex that is complementary to a nucleotide sequence that is complementary to the nucleotide sequence.
  • stringent hybridization conditions include 6M urea, 0.4% SDS, 0.5 ⁇ SSC, or 0.1% SDS (60 ° C., 0.3 mol NaCl, 0.03M sodium citrate). ) Hybridization conditions or stringent hybridization conditions equivalent to these. Under conditions of higher stringency, for example, conditions of 6M urea, 0.4% SDS, 0.1 ⁇ SSC, nucleotide sequences with higher homology can be identified.
  • nucleotide sequences shown in (s), (w) and (k) are nucleotide sequences having at least about 80% sequence identity with the nucleotide sequences described in (p), (t) and (h), respectively. It is. Furthermore, the nucleotide sequences shown in (s), (w) and (k) have at least about 85% sequence identity to the nucleotide sequences described in (p), (t) and (h), respectively.
  • the nucleotide sequence is preferably about 90% or more, and more preferably about 91% or more, 92% or more, 93% or more, 94% or more, 95% or more. 96% or more, 97% or more, 98% or more, 99% or more nucleotide sequence.
  • the gene expression control sequence (R) in the nucleic acid according to the present invention may further include other gene expression regulatory sequences in addition to the nucleotide sequences (X) and (Y).
  • examples of such other gene expression regulatory sequences include further transcription regulatory sequences such as a lac operator, translation regulatory sequences such as a ribosome binding sequence (Shine-Dalgarno sequence; SD sequence), and the like.
  • the nucleic acid according to the present invention is provided in the form of an expression cassette, an expression vector or the like, for example, the SD sequence shown in (3-1) to (3-6) below may be included.
  • the position of the SD array is not particularly limited as long as the desired effect of the present invention can be obtained.
  • the SD sequence should be present between the 3 ′ end of the nucleotide sequence (Y) and the start codon.
  • the SD sequence is linked directly or indirectly to the 3 ′ end of the nucleotide sequence (Y).
  • the SD sequence shown in (3-1) is an SD sequence combined with PtacI and PtacII, and as shown in the following examples, ensures strict gene expression control of the target gene using arabinose.
  • a high gene expression level at the protein expression level can be promised. More specifically, the embodiments according to the above (p) to (s), the embodiments according to (t) to (w), the embodiments according to (h) to (k), and the modifications described above.
  • nucleotide sequence defined in each of (p), (t) and (h) is directly ligated to the 3 ′ end of the nucleotide sequence and the sequence shown in (4-1) below.
  • the nucleic acid is a terminator (transcription termination) that can function in bacteria (especially Escherichia coli and / or coryneform bacteria). Signal) (eg, T rnb , T GroEL , T trp , T T7 ).
  • the position of the terminator is not particularly limited as long as the desired effect of the present invention is obtained.
  • the terminator can be directly or indirectly linked to the 3 ′ end of the nucleotide sequence (Y) or the SD sequence.
  • a terminator can be directly or indirectly linked to the 3 ′ end of the cloning site.
  • a terminator can be linked directly or indirectly to the 3 ′ end of the target gene coding region.
  • nucleic acid according to the present invention may optionally further comprise a nucleotide sequence encoding at least one of araE protein and araR protein. This is because if the araE protein and araR protein are forcibly expressed in the microbial cells, the certainty and strictness of gene expression control by addition of arabinose can be ensured.
  • nucleotide sequence (Y) is derived from the AraR binding sequence present in the genomic DNA of Corynebacterium glutamicum as described above, coryneform bacteria, preferably Corynebacterium, more preferably Corynebacterium It is preferably a nucleic acid (DNA) into which at least one of the araE gene coding sequence and the araR gene coding sequence present in the genomic DNA of bacteria glutamicum is inserted.
  • nucleic acid according to the present invention may further comprise one or more nucleotide sequences selected from the group consisting of at least one replication origin, a selection marker gene, a cloning site and a restriction enzyme recognition site.
  • the origin of replication and selectable marker gene may be functional in general bacteria or a plurality of types of bacteria, or may be functional in a specific bacterial species. For example, what contains any one or more of the following is mentioned.
  • an origin of replication and / or a selectable marker gene coding sequence that can function only in coryneform bacteria (2) an origin of replication and / or a selectable marker gene coding sequence that is functional only in Escherichia (eg, Escherichia coli); and (3) an origin of replication that is functional in both coryneform bacteria and Escherichia.
  • Selectable marker gene coding sequence (1) an origin of replication and / or a selectable marker gene coding sequence that can function only in coryneform bacteria; (2) an origin of replication and / or a selectable marker gene coding sequence that is functional only in Escherichia (eg, Escherichia coli); and (3) an origin of replication that is functional in both coryneform bacteria and Escherichia.
  • nucleic acid according to the present invention can be provided as a plasmid capable of autonomous replication in bacteria including coryneform bacteria and Escherichia bacteria.
  • nucleic acids according to the present invention can be provided as expression vectors.
  • the nucleic acid according to the present invention is, for example, provided in the form of a shuttle expression vector that can autonomously replicate in both coryneform bacteria and Escherichia bacteria, and that can express a target gene in coryneform bacteria. There may be.
  • the nucleic acid according to the present invention may further include a nucleotide sequence (Z) linked directly or indirectly to the 3 'end of the nucleotide sequence (Y) and encoding the target gene.
  • the nucleotide sequence (Z) is not an essential component in the nucleic acid according to the present invention.
  • nucleic acid fragment for use in controlling expression of a target gene in coryneform bacteria, A nucleic acid fragment comprising the nucleotide sequence (Y) described in the following (a) or (b): (A) the nucleotide sequence set forth in any one of SEQ ID NOs: 5 to 9, 11, and 12 and SEQ ID NOs: 17 and 18; (B) a nucleotide sequence in which one or more bases have been deleted, substituted or added in the nucleotide sequence described in (a) above, However, When the nucleic acid is RNA, thymine (t) in the nucleotide sequence shall be read as uracil (u), The nucleotide sequence shown in each of SEQ ID NOs: 10, 20, and 21 as the nucleotide sequence (Y) is excluded, and the nucleotide sequence (Y) satisfies the following condition (II): Condition (I
  • the promoter activity of PtacI is preferably suppressed to 1/8 or less, more preferably 1/9 or less, 1/10 or less, 1/15 or less, 1/20 or less, 1/25 or less, and more Preferably 1/30 or less, 1/35 or less, 1/40 or less, 1/45 or less, particularly preferably 1/50 or less, 1/55 or less, 1/60 or less, 1/65 or less, 1/70 or less, 1/80 or less, 1/90 or less, 1/100 or less, or 1/150 or less.
  • the nucleotide sequences shown in SEQ ID NOs: 10, 20, and 21 are natural AraR binding sequences (BS E1 , BS E2 , BS B ) (Non-Patent Document 6).
  • nucleic acid according to the fifth aspect are as described for the nucleic acid according to the first aspect. As long as it does not occur, it can also be adopted in the nucleic acid according to the fifth aspect, and various combinations of these embodiments or features are disclosed herein as embodiments that can be adopted in the nucleic acid according to the fifth aspect. It is. Furthermore, the nucleic acid according to the fifth aspect can also be used in the following further aspects.
  • a bacterium into which the nucleic acid according to the present invention has been introduced is not particularly limited, and examples include Escherichia and the above-mentioned coryneform bacteria (preferably Corynebacterium).
  • the bacterium introduced with the nucleic acid according to the present invention is one in which a nucleic acid containing at least one nucleotide sequence encoding each of the araE protein and araR protein derived from coryneform bacteria is incorporated into genomic DNA so that these proteins can be expressed. May be.
  • the nucleic acid according to the present invention introduced into the bacterium specifically has a nucleotide sequence (Z) encoding a target gene 3 ′ downstream of the gene expression control sequence (R) (nucleotide sequence (Y)). It can be concatenated. This is because when the araE protein and the araR protein are forcibly expressed in the microbial cells, the above-described expression control of the target gene by the addition of arabinose is more reliably and efficiently realized.
  • the nucleic acid according to the present invention similarly encodes araE protein and araR protein, respectively.
  • the at least one nucleotide sequence may not be included, or the at least one nucleotide sequence may be included.
  • a method for expressing a target gene which comprises expressing the target gene by exposing the bacterium introduced with the nucleic acid of the present invention to arabinose or an analog thereof.
  • a method for producing a target substance which comprises expressing a target gene by exposing the bacterium introduced with the nucleic acid of the present invention to arabinose or an analog thereof.
  • a specific embodiment of “exposing a bacterium to arabinose or an analog thereof” in the method of the present invention is not particularly limited as long as promoter activity is induced.
  • the step of culturing the bacterium of the present invention The bacterium may be exposed to arabinose by adding arabinose to the medium.
  • the concentration of arabinose or an analog thereof in the medium is not particularly limited as long as the promoter activity is induced.
  • it is 0.001% or more, preferably 0.005% or more, more preferably 0.00. 007%, still more preferably 0.008% or more, 0.009% or more, particularly preferably 0.01% or more, 0.015% or more, 0.018% or more.
  • the upper limit of the concentration of arabinose or an analog thereof is not particularly limited as long as promoter activity is induced. However, in consideration of cost, it is 5% or less, preferably 3% or less, more preferably 2.5. % Or less.
  • the numerical range in which these upper limit value and lower limit value are arbitrarily combined is disclosed herein as a range of the concentration of arabinose or an analog thereof upon induction of promoter activity, and in a specific embodiment of the present invention. Can be employed.
  • arabinose is more specifically L-arabinose.
  • an arabinose analog (“analog thereof”) is the same gene expression control as arabinose in the presence of the gene control sequence (R) (nucleotide sequence (Y)) in the nucleic acid according to the present invention.
  • R gene control sequence
  • Y nucleotide sequence
  • the induction time of promoter activity by addition of arabinose may be adjusted as appropriate so as to obtain expression of the desired promoter activity, and is not particularly limited.
  • the time can be 5 to 48 hours, preferably 1 to 24 hours, more preferably 1 to 12 hours, and 1 to 6 hours.
  • a sufficient increase in promoter activity and the expression level of the target gene can be ensured in a very short time. Therefore, biotechnology excellent in efficiency / productivity and economy can be secured. Process can be realized.
  • a protein that is the translation product of the target gene may be produced as the target substance, or the protein that is the translation product of the target gene is contained in the microbial cells.
  • a substance for example, a metabolite
  • examples of the protein that is the translation product of the target gene include an enzyme, and the target substance can be produced by progressing metabolism by the recombinant enzyme expressed in the microbial cells.
  • the nucleic acid of the present invention having the above-described configuration includes, for example, genomic DNA or plasmid DNA possessed by various bacteria including coryneform bacteria and Escherichia, and genomic nucleic acids possessed by various bacteriophages capable of infecting these bacteria, and artificially designed It can be produced based on genetic engineering techniques / molecular biological techniques including various cloning techniques and mutagenesis techniques using resources such as nucleic acid fragments having the nucleotide sequences prepared as materials.
  • genetic engineering techniques / molecular biological techniques including various cloning techniques and mutation introduction techniques see, for example, Molecular Cloning: A Laboratory Manual, Fourth Edition (3-Volume Set), Cold Spring Harbor Laboratory Pr, etc.
  • nucleic acid according to the present invention can be produced using such a known technique with reference to the disclosure of the present specification.
  • part or all of the nucleic acid according to the present invention may be produced by chemical synthesis.
  • the bacterium according to the present invention can also be prepared by referring to the disclosure of the present specification and various genetic engineering techniques / molecular biological techniques. Examples of nucleic acids / bacteria according to the present invention, methods for producing them, and methods according to the present invention are shown in the following examples.
  • Test Example 1 In Test Example 1, an araE gene, an araR gene, and an expression vector into which a gene regulatory sequence (R) and a target gene (reporter gene) according to the present invention were introduced were introduced into a coryneform bacterium, and the target gene (reporter gene) The example which controlled the expression of is shown. Details of the test procedure are shown below.
  • K a nR shown in SEQ ID NO: 63, 64 and 65 the nucleotide sequence of the amplified region by PCR for pUCori and PCG1ori.
  • pCG1 is extracted from the above strain according to a conventional method.
  • PldhA-rrnBter / K a nR / pUCori / pCG1ori are arranged in this order.
  • the amplification of the PldhA fragment used Corynebacterium glutamicum ATCC13032 strain genomic DNA as a template
  • the amplification of the rrnBter fragment used the plasmid vector pFLAG-CTC (Sigma) as a template.
  • the nucleotide sequences of these amplified fragments are shown in SEQ ID NOs: 66 and 67, respectively.
  • pGEK020 (PldhA-lacZ-rrnBter / KanR / pUCori / pCG1ori)
  • pGEK008 was cleaved with NdeI and BamHI to obtain a linear DNA fragment.
  • This linear DNA fragment was cleaved at the restriction enzyme recognition site in the region between PldhA and rrnBter.
  • a LacZ gene fragment (LacZ fragment) was separately amplified by PCR using a template described later. At this time, an NdeI site and a BamHI site were inserted into the primer pair as adapter sequences, respectively, and the amplified LacZ fragment was similarly cleaved with NdeI and BamHI.
  • pGEK008 cleavage fragment and the LacZ fragment were ligated in a circular shape to obtain a plasmid vector pGEK020.
  • PldhA-lacZ-rrnBter / K a nR / pUCori / pCG1ori are arranged in this order.
  • a plasmid vector owned by the applicant was used as a template.
  • the LacZ gene region amplified in the plasmid vector owned by the applicant is derived from pSV- ⁇ -Galactosidase Control Vector (Promega) and contains the same nucleotide sequence as the LacZ gene coding region of the vector.
  • a commercially available plasmid containing the E. coli genomic DNA or the lacZ gene coding region may be obtained and the LacZ gene coding region may be cloned.
  • the nucleotide sequence of the amplified LacZ gene fragment is shown in SEQ ID NO: 68.
  • pGEK030 (MCS-lacZ-rrnBter / KanR / pUCori / pCG1ori) pGEK020 was cleaved with NdeI and NotI to obtain a linear DNA fragment from which the region from pCG1ori to PldhA was removed.
  • pGEK094 (Ptac-lacZ-rrnBter / KanR / pUCori / pCG1ori)
  • a derived plasmid obtained by introducing another element into MCS of pGEK020 was used as a material.
  • the difference between the derived plasmid and GEK020 is only whether or not another element is inserted into MCS, and this other element is deleted by treating the derived plasmid with KpnI and NdeI as follows when preparing pGEK094. Is.
  • pGEK094 constructed in this procedure (5) is equivalent to the one in which the Ptac fragment described below is inserted between the KpnI site and the NdeI site in the MCS of pGEK020. Therefore, the details of the steps for preparing the derivative plasmid actually used for the construction of pGEK094 are omitted. Furthermore, the Ptac fragment was obtained by amplifying by PCR using the plasmid vector owned by the applicant as a template. The Ptac region in the plasmid vector is derived from the commercially available plasmid vector pFLAG-CTC (Sigma). Is.
  • the Ptac fragment can be amplified using pFLAG-CTC (Sigma) as a template.
  • the nucleotide sequence of the actually amplified Ptac fragment is shown in SEQ ID NO: 72. Then, the linear DNA fragment obtained by cleaving the derived plasmid with KpnI and NdeI and the amplified Ptac fragment were ligated into a circular shape using Gibson Assembly Cloning Kit to obtain pGEK094.
  • the nucleotide sequence of the amplified T7 terminator fragment is shown in SEQ ID NO: 73.
  • the elements of T7Uter-T7ter-rrnBT1ter are linked in this order, and the order of the elements differs from that disclosed in the above document.
  • the linker sequence present between each element is also different from that disclosed in the above document.
  • the lacI gene was inserted in the process of preparing various vectors by the inventor, but was finally removed in the vector according to the present invention as described below. Therefore, the nucleotide sequence information of the amplified lacI gene coding region is omitted.
  • the above pGEK094 was cleaved with KpnI and AgeI, and the obtained digested product was ligated with the amplified lacI gene fragment and T7 terminator fragment using GibsonbAssembly Cloning Kit to obtain a plasmid vector pGEK122.
  • pGEK122 was cleaved with the restriction enzyme XbaI, and the obtained digested product, and the PdapA fragment and the araE fragment were ligated to form a circle with the In-Fusion HD Cloning Kit.
  • the vector thus prepared was named pGE651.
  • Construction of pGE677 (PdapA-araE-rrnBT1ter-T7ter-T7Uter-lacI / Ptac-lacZ-rrnBter / KanR / pSC101 / pCG1ori)
  • pGE677 A vector exchanged with the Escherichia coli replication origin pSC101 ori (low copy number) contained in the plasmid vector was prepared and named pGE677.
  • the pSC101ori region in the plasmid vector owned by the applicant is derived from the plasmid vector pMW119 (purchased from Takara Bio, currently available from Nippon Gene), and contains the same nucleotide sequence as the pSC101ori region of the vector.
  • the nucleotide sequence of the pSC101ori region containing the restriction enzymes NcoI and BamHI at both ends is shown in SEQ ID NO: 76.
  • the araR gene fragment was amplified by PCR.
  • the nucleotide sequence of the amplified araR gene coding region is shown in SEQ ID NO: 78.
  • a fragment in which an AraR binding sequence was linked to Ptac was amplified by PCR using pGEK094 as a template. The nucleotide sequence of this fragment is shown in SEQ ID NO: 79.
  • the pGE677 obtained in (8) above is cleaved with restriction enzymes AgeI and NdeI to obtain a linear DNA fragment from which the T7Uter and LacI regions have been removed, and this DNA fragment, as well as the PdapA fragment, araR gene fragment and Ptac
  • the fragment having the araR binding sequence linked to was ligated into a circular shape by Gibson Assembly Cloning Kit to prepare pGE728-1.
  • the structure of pGE728-1 is shown in FIG. 1 (a), and its nucleotide sequence is shown in SEQ ID NO: 80.
  • plasmid vectors pGE728-2 to pGE728-12 having a predetermined mutation in the AraR binding sequence (AraRBS) with respect to pGE728-1 were obtained.
  • the AraR binding sequences in pGE728-1 to pGE728-12 are shown in Table 1 below. As shown in 1-12.
  • LacZ assay expression of ⁇ -galactosidase
  • the pGE728-1 to pGE728-12 prepared as described above were evaluated for the possibility of gene expression control according to the ⁇ -galactosidase gene (LacZ) reporter assay procedure described in the above embodiment.
  • the sample was sampled after culturing for 5 hours, and the activity of LacZ was measured for the obtained sample. [result] Table 1 below shows the results of Test Example 1.
  • Sample No. Sample Nos. 9 to 12 were combined with AraR binding sequences (SEQ ID NOs: 5 to 12) each having a predetermined nucleotide sequence in Ptac.
  • SEQ ID NOs: 5 to 12 AraR binding sequences
  • sample No. 2, 3, 4, and 6 have relatively high induction efficiency of promoter activity, and in particular, sample Nos. Obtained by combining SEQ ID NOs: 2 and 3 with Ptac. 2 and 3 showed that the promoter activity increased more than 80-fold when arabinose was added compared to the case where arabinose was not added.
  • the promoter activity (LacZ expression) can be remarkably suppressed, and at the same time, the promoter activity increases particularly remarkably by adding arabinose, and the target gene product in an active form is increased in a large amount. It was shown that it can be expressed. Therefore, the form in which the nucleotide sequence of SEQ ID NO: 2 or 3 is combined with Ptac enables particularly strict control of gene expression (switch on / off) and is particularly high when the gene expression is switched on. It can be said that it is a particularly preferred embodiment in the present invention in that the expression level can be secured. Thus, according to the predetermined sequence configuration that can be possessed by the nucleic acid according to the present invention, it was shown that efficient and precise gene expression control by adding / adding arabinose becomes possible.
  • Table 2 shows the results of Test Example 2.
  • pGE728-2 and pGEE728-3 confirmed to show excellent gene expression control ability in Test Example 1 were also used in this test example.
  • the promoter activity ratios were 94.1 and 98.9, respectively, and it was confirmed again that the gene expression control ability was excellent.
  • pGE728-13 shows a promoter activity ratio of 162, and the above-mentioned pGE728-2 and pGEE728-3 The value of the promoter activity ratio was far exceeded, and it was confirmed that the gene expression control ability was extremely excellent.
  • pGE728-14 newly constructed in this test example has sufficient gene-regulated expression ability although the promoter activity ratio is a relatively low value of 14.8. .
  • Test Example 3 the expression of the target gene (reporter gene) was controlled using an expression vector containing the gene control sequence according to the present invention using a coryneform bacterium in which the araE gene and the araR gene were incorporated into the genome instead of a plasmid vector. An example is shown. Details of the test procedure are shown below. (1) Preparation of expression plasmid vector pGE728 was cleaved with restriction enzymes KpnI and XbaI to obtain a DNA fragment from which the araE coding region and the araR coding region were removed.
  • the DNA fragment was blunted using T4 DNA polymerase (Takara Bio), subjected to terminal phosphorylation with T4 polynucleotide kinase (Takara Bio), and then ligated with T4 DNA ligase (NEB) for circularization.
  • T4 DNA polymerase Takara Bio
  • Takara Bio T4 polynucleotide kinase
  • NEB T4 DNA ligase
  • the resulting plasmid was named pGE716.
  • the structure of pGE716 is shown in FIG. 1 (b), and its nucleotide sequence is shown in SEQ ID NO: 81.
  • a plasmid vector pGE825 was obtained by circularly ligating the amplified fragment containing the upstream and downstream regions of the tnp1a and the linear DNA fragment of the pGE285 using the In-Fusion HD Cloning Kit.
  • a DNA fragment in which Ptac, the araE gene coding region present in the genome of Corynebacterium glutamicum ATCC 31831 strain, and the rrnBter terminator were ligated in this order was used as a plasmid vector owned by the applicant. Amplified by PCR as a template. The nucleotide sequence of the amplified Ptac-araE-rrnBter fragment is shown in SEQ ID NO: 87.
  • the plasmid vector pGE837 was obtained by circularly ligating the linear DNA fragment of pGE825 and the amplified Ptac-araE-rrnBter fragment using In-Fusion HD Cloning Kit.
  • the structure of pGE837 is shown in FIG. 2 (a), and its nucleotide sequence is shown in SEQ ID NO: 88.
  • a fragment containing the region of PldhA-lacZ-rrnBter (PldhA-lacZ-rrnBter fragment) was amplified by the PCR method using pGEK020 constructed in Test Example 1 as a template.
  • This fragment was inserted in the process of making various vectors in the inventor, but as described below, in the pAra1 finally used for introducing the araR gene into the coryneform bacterial genome, PldhA and As a result, the lacZ region is removed. Therefore, information on the nucleotide sequence of the amplified PldhA-lacZ-rrnBter fragment is omitted.
  • pGE209 linear DNA, Ptac fragment, araR gene fragment, and In-Fusion HD Cloning Kit were used for ligation so as to form a circular shape, thereby obtaining a plasmid vector pAra1.
  • the structure of pAra1 is shown in FIG. 2 (b), and its nucleotide sequence is shown in SEQ ID NO: 93.
  • Test Example 3 is different from Test Example 1 in the following points. That is, in Test Example 1, as a plasmid vector to be introduced into the ATCC 13032 strain, one encoding the araE gene and the araR gene in addition to the gene expression control sequence (R) and reporter gene (LacZ gene) of the present invention was used. . On the other hand, in Test Example 3, AIS1 strain (derived from ATCC13032 strain) introduced so that the araE gene and araR gene were forcibly expressed in the genomic DNA in advance was used, and the araE gene was used as a plasmid vector to be introduced into the strain. And pGE716 from which the araR gene was removed.
  • AIS1 / pGE716-3 and AIS1 / pGE716-3 are samples derived from different transformant colonies, respectively. Focusing on the fold (induction efficiency) of the promoter activity, AIS / p716-3 shows 67 times and AIS / p716-4 shows 160 times. According to the gene expression control sequence of the present invention, arabinose is effective. It was shown that the expression of the target gene can be controlled.
  • Test Example 4 Influence of arabinose concentration / induction time
  • pGE716 was electroporated into strain AIS1 and selected with kanamycin to obtain a transformant.
  • a sample without arabinose added was also prepared as a comparative control.
  • the above samples were cultured, and after 4 hours, 20 hours, and 24 hours, the culture medium with and without arabinose induction was sampled and the promoter activity was measured by LacZ assay.
  • the procedure of the LacZ assay and the composition of the liquid medium are as described in Test Example 3.
  • DNA fragments each having the nucleotide sequence shown in SEQ ID NOs: 96 to 102 were obtained. These DNA fragments each contain the nucleotide sequence according to SEQ ID NOs: 96 to 102 shown as a preferred embodiment of the nucleic acid according to the present invention in the section of the above embodiment.
  • PGEK107 obtained in (2) above is cleaved with restriction enzymes KpnI and NdeI, and each promoter / AraR binding sequence fragment is circularized with the In-Fusion HD Cloning Kit in the resulting vector fragment. Connected.
  • the plasmids thus obtained were designated as pAra24, pAra25, pAra26, pAra27, pAra28, pAra29, and pAra30, respectively.
  • each plasmid was electroporated in strain AIS1 and selected with kanamycin to obtain transformants.
  • the promoter site is not limited to the tac promoter. It has been proved that excellent gene regulation ability can be exhibited even when various other promoters including a general promoter and an inducible promoter are employed.
  • Comparative Test Example 1 is a comparative example in which control of the target gene was attempted using the promoter region of the araE gene that is naturally present in the genome of Corynebacterium glutamicum ATCC 31831 instead of the control sequence according to the present invention. Details of the test procedure are shown below.
  • the activity value with arabinose induction (that is, arabinose added) is lower than the activity value without arabinose induction (that is, without arabinose added), and arabinose cannot control gene expression. It was shown that.
  • the nucleotide sequence used as the gene regulatory sequence in this Comparative Test Example 1 is inherently present on the genomic DNA of Corynebacterium glutamicum ATCC 31831 as shown in FIG. 5 and includes the natural AraR binding sequence. It is a waste. That is, even when a promoter region unique to the coryneform bacterium as shown in Non-Patent Document 6 is used, as shown in the results of Table 2, the gene expression control achieved by the predetermined nucleotide sequence of the present invention is not possible. It was possible and did not even show promoter activity as a gene expression system in the first place.
  • nucleotide sequence information such as various gene coding regions, promoter regions, terminator regions, and replication origins amplified by the PCR method during the construction of various plasmid vectors and the like is disclosed in the sequence listing. Therefore, the sequence information of the primers used for PCR amplification was omitted.
  • the person skilled in the art refers to the construction of the plasmid vector or the like used in the above test examples or the present invention by referring to the information of each nucleotide sequence disclosed in the sequence listing or the information of the nucleotide sequence present in the gene bank etc. Nucleic acids and the like can be appropriately produced.
  • various gene coding regions, promoter regions, terminator regions, replication origins, etc. may be cloned using the In-Fusion HD Cloning Kit or Gibson Assembly Cloning Kit as described above.
  • the primer pair used in PCR amplification is supplemented by adding an appropriate adapter sequence to the 5 ′ end of the forward / reverse primer according to the instructions of the cloning kit.
  • the present invention has high industrial applicability in the fields of biotechnology, production of substances such as chemical substances and biological materials.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne : un acide nucléique qui comprend une séquence de régulation de l'expression des gènes qui peut réguler l'expression d'un gène cible dans des bactéries corynéformes ; des bactéries dans lesquelles l'acide nucléique a été introduit ; un procédé d'utilisation des bactéries pour exprimer le gène cible ; et un procédé d'utilisation des bactéries pour produire une substance. Au moins une partie de l'acide nucléique comporte une séquence de promoteur arbitraire et une séquence de liaison à l'AraR qui est spécifiée par une séquence nucléotidique prescrite. Lorsque l'acide nucléique est introduit dans des bactéries corynéformes, l'activité de promoteur de la séquence de promoteur est supprimée tant que l'arabinose n'est pas ajouté, et est induite lorsque l'arabinose est ajouté.
PCT/JP2019/004378 2018-02-08 2019-02-07 Acide nucléique comprenant une séquence de régulation de l'expression des gènes dépendant de l'arabinose Ceased WO2019156152A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2019570794A JP7374475B2 (ja) 2018-02-08 2019-02-07 アラビノース依存型遺伝子発現制御配列を含む核酸

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2018021453 2018-02-08
JP2018-021453 2018-02-08

Publications (1)

Publication Number Publication Date
WO2019156152A1 true WO2019156152A1 (fr) 2019-08-15

Family

ID=67548309

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2019/004378 Ceased WO2019156152A1 (fr) 2018-02-08 2019-02-07 Acide nucléique comprenant une séquence de régulation de l'expression des gènes dépendant de l'arabinose

Country Status (2)

Country Link
JP (1) JP7374475B2 (fr)
WO (1) WO2019156152A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008001597A1 (fr) * 2006-06-30 2008-01-03 Ajinomoto Co., Inc. Procédé de production d'une protéine
JP2010535028A (ja) * 2008-04-10 2010-11-18 コリア アドバンスド インスティチュート オブ サイエンス アンド テクノロジィ プトレッシン高生成能を有する変異微生物及びこれを用いたプトレッシンの製造方法
WO2013069634A1 (fr) * 2011-11-11 2013-05-16 味の素株式会社 Procédé de production d'une substance cible par fermentation

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008001597A1 (fr) * 2006-06-30 2008-01-03 Ajinomoto Co., Inc. Procédé de production d'une protéine
JP2010535028A (ja) * 2008-04-10 2010-11-18 コリア アドバンスド インスティチュート オブ サイエンス アンド テクノロジィ プトレッシン高生成能を有する変異微生物及びこれを用いたプトレッシンの製造方法
WO2013069634A1 (fr) * 2011-11-11 2013-05-16 味の素株式会社 Procédé de production d'une substance cible par fermentation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KUGE, T. ET AL.: "AraR, an L-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions", JOURNAL OF BACTERIOLOGY, vol. 197, no. 24, 28 September 2015 (2015-09-28) - December 2015 (2015-12-01), pages 3788 - 3796, XP055629746 *
KUGE, T. ET AL.: "The LacI-Type Transcriptional Regulator AraR Acts as an L-Arabinose-Responsive Repressor of L-Arabinose Utilization Genes in Corynebacterium glutamicum ATCC 31831", JOURNAL OF BACTERIOLOGY, vol. 196, no. 12, 4 April 2014 (2014-04-04) - June 2014 (2014-06-01), pages 2242 - 2254, XP055629741 *
KUGE, TAKAYUKI ET AL.: "Arabinose response Control mechanism by transcription factor AraR in coryneform bacteria", PROGRAMS OF THE 2015 CONFERENCE OF JSBBA, 25 February 2015 (2015-02-25) *
KUGE, TAKAYUKI: "Engineering of Corynebacterium glutamicum for arabinoxylan utilization", DOCTORAL DISSERTATION OF THE GRADUATE SCHOOL OF BIOLOGICAL SCIENCES OF NARA INSTITUTE OF SCIENCE AND TECHNOLOGY, March 2017 (2017-03-01), pages 1 - 120, Retrieved from the Internet <URL:https://library.naist.jp/mylimedio/dllimedio/showpdf2.cgi/DLPDFR013620_P1-120> [retrieved on 20190423] *

Also Published As

Publication number Publication date
JP7374475B2 (ja) 2023-11-07
JPWO2019156152A1 (ja) 2021-02-12

Similar Documents

Publication Publication Date Title
AU2016316027B2 (en) Systems and methods for selection of gRNA targeting strands for Cas9 localization
JPH0970291A (ja) 人工トランスポゾンを用いた遺伝子増幅方法
BR112019004161B1 (pt) Molécula de ácido nucleico que possui uma atividade promotora, cassete de expressão gênica, vetor recombinante, micro-organismo recombinante do gênero corynebacterium e método de produzir um produto-alvo
JP6335196B2 (ja) アコニターゼ遺伝子および／またはその調節エレメントの改変により、ｌ−リジンを生産する方法
JPS58192900A (ja) 複合プラスミド
BRPI9909409B1 (pt) processos para produzir um ácido l-glutâmico
JP2023109990A (ja) プリンヌクレオチドを生産する微生物及びそれを用いたプリンヌクレオチドの生産方法
Hashiro et al. High copy number mutants derived from Corynebacterium glutamicum cryptic plasmid pAM330 and copy number control
JP2022547466A (ja) 新規なプロモーター及びそれを用いた標的物質生産方法
JP7789070B2 (ja) 遺伝子ｂｂｄ２９＿０９５２５を改造したｌ－グルタミン酸生産組換え菌株及びその構築方法と応用
JP7374475B2 (ja) アラビノース依存型遺伝子発現制御配列を含む核酸
KR101959063B1 (ko) L-로이신 생산능이 개선된 변이 균주 및 이를 이용한 l-로이신의 제조 방법
PL222067B1 (pl) Kaseta ekspresyjna, zastosowanie kasety ekspresyjnej, wektor ekspresyjny, komórka prokariotycznego gospodarza zawierająca wektor ekspresyjny, szczep bakteryjny oraz sposób wytwarzania polipeptydu
CN113278620B (zh) 一种突变的高渗诱导型启动子PproP及其应用
JP7766704B2 (ja) Ｌ－リシン生産能が向上したコリネバクテリウム・グルタミカム変異株及びそれを用いたｌ－リシン生産方法
CN115927325B (zh) 柠檬酸合酶启动子突变体及其应用
EP4689136A1 (fr) Hôtes et phagémides de production d&#39;adncss modifiés
JP7809708B2 (ja) 遺伝子ｂｂｄ２９＿１１２６５を改造してｌ－グルタミン酸を生産する組換え菌株及びその構築方法と応用
EP4293115A1 (fr) Polynucléotide ayant une activité de promoteur et son utilisation dans la production de composés cibles
CN113201538B (zh) 具有启动子活性的多核苷酸及其在生产目标化合物中的用途
JP7805404B2 (ja) 恒常発現用新規プロモーター、これを含む目的タンパク質発現システムおよびこれを用いてアルロースを製造する方法。
KR102869970B1 (ko) 변이된 rbs 염기 서열을 포함하는 폴리뉴클리오티드, 상기 폴리뉴클레오티드를 포함하는 미생물, 및 이를 이용한 글리신 생산 방법
JP7620123B2 (ja) Ｌ－リシン生産能が向上したコリネバクテリウム・グルタミカム変異株及びそれを用いたｌ－リシン生産方法
AU2021442398B2 (en) Corynebacterium glutamicum variant having improved l-lysine production ability, and method for producing l-lysine using same
WO2008026698A1 (fr) Procédé de production d&#39;acide l-glutamique

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19751311

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2019570794

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19751311

Country of ref document: EP

Kind code of ref document: A1